X-inactivation is a mammalian mechanism to compensate for dosage differences between females with two X-chromosomes and males with one X chromosome. X-inactivation requires the Xist gene, a gene encoding a large structural RNA that coats the inactive X-chromosome and interacts with the nuclear matrix. This research plan is based on the hypothesis that Xist RNA plays a structural and functional role in forming the heterochromatic X-chromosome and occurs within a ribonucleoprotein (RNP) complex. To understand how Xist functions and how heterochromatin is constructed in X-inactivation, I will identify factors which interact with Xist RNA, focusing specifically on protein components of the RNP. To maximize the chances of isolating Xist RNA interacting proteins, two complementary strategies will be taken in parallel: 1) The yeast three hybrid system will be used to screen for interacting proteins using Xist RNA fragments as "bait". This novel genetic approach takes advantage of the existing expertise and reagents in Dr. Lee's laboratory. 2) A biochemical approach based on affinity chromatography. Xist RNA-containing complexes will be purified by utilizing the ability of Xist RNA to bind antisense Xist cDNA immobilized on magnetic beads. Since Xist RNA is large, it is likely to bind many factors in vivo. Using both a genetic and a biochemical approach is important because their differing methodologies may yield the identification of different interacting partners for Xist RNA.